Electrophoresis is a process for separating a mixture of particles into separate groups of similar particles using the movement of ions in an electric field (drift currents). In capillary electrophoresis, a small tube (sometimes called a “capillary”) with an inner diameter that is small enough to avoid turbulent flow is filled with an aqueous solution. A small amount of a mixture to be analyzed is provided at one end of the tube. An indicator stain or fluorescent marker may be included in the mixture to assist in resolving groups of particles after separation has occurred. A voltage, typically in the low tens of thousands of volts, is placed across the tube. Positive ions begin to move toward the negative electrode, and negative ions begin to move toward the positive electrode creating a current in the low tens of milliamps. The speed at which the ions move is determined by the size of the voltage (electric field), the charge on the ion, and the resistance to movement through the solution in the tube. Since different particles in the mixture have different resistances to movement, each type of particle moves at a different rate, and thus, the different particles begin to separate due to their differing resistances to movement. Over time, different and distinct particle groups form along the length of the tube. A group of the same particles, e.g., the same molecules, often appear in the shape of a plate or a band. Ultimately, electrophoresis is a stable way to separate a heterogeneous solution of particles into distinct individual particle groups.
FIG. 1 illustrates this electrophoresis process and shows a mixture of particles 12 introduced at one end of a capillary tube 10. As time progresses, the mixture separates into two distinct groups or groups of particles with one group 12A including one type of particle and the other group 12B including a second different type of particle. Each group is shown as a plate or band. Because one of these groups is faster than the other group, a separation distance develops. Once separated by a sufficient distance, the two groups may be identified and distinguished.
FIG. 2 illustrates an electrophoresis setup that includes probe light focused by a lens 14 through the tube 10 onto a stationary optical detector 16. The optical detector 16 detects each group as it crosses a detector zone that includes probe light and a focusing lens. Typically, only one location along the tube 10 is monitored. An output of a capillary electrophoresis instrument coupled to the detector 16 may be a plot of detected material, e.g., by UV fluorescence or absorbance, as a function of time. FIG. 3 is an illustration of an example capillary electrophoresis plot with peaks arriving over the course of 1 to 10 minutes.
In the case of fast moving molecules, gel electrophoresis is implemented to increase the resistance to movement for the molecules. Similar to the first example, groups of particles migrate down a tube under a voltage and the gel ensures separation can be achieved before the particles reach the end of the tube by supplying a higher resistance to movement. A drawback of gel type and other known electrophoresis techniques is that they are time consuming. A significant amount of time must be waited before each group of particles passes the detection location, and this time period is even longer when a higher resistive transportation medium is used in the tube. For example, FIG. 4 shows a spatial distribution of three different groups of particles at 3 minutes into the test. It would be desirable to identify the individual groups as they separate and move down the capillary tube before reaching the single detection location in order to speed up the process.
In addition to long processing times, known technology suffers from a limited detection region. As seen in FIG. 2, a typical setup consists of a single monitoring location. This is due to the complexity of setting up additional detection points as each requires a probe light, focusing lens, and detection system. This location must be monitored continuously, and without interruption, as particles that pass that detection location when the system is not monitoring the location will have no means of being detected as they progress in a single direction through the tube. This causes multiplexing of several sample tubes to be impractical, as a sample may be missed as another tube is being monitored. Another problem is that single point scanning approach requires that the length of the capillary tube be determined before the contents and separation rate of the various particles are known.
The inner diameter of the capillary tube is also restricted in current techniques. As the inner diameter decreases, optical detection becomes more difficult because the path through the solution becomes smaller as well. Reduced path length means a lower number of particles will exist in the volume requiring higher concentrations for successful detection. It is desired in the technique to use smaller tubes. As the inner diameter decreases, there is less current carried by the solution, and thus, less heat dissipation. Further, whatever heat is generated is more quickly moved out of the solution and into the tube wall. Lower temperature means less thermal diffusion and better resolution of the particle groups. The ability to use smaller capillary inner diameters means that high separation rates (due to higher voltages) can be achieved with better resolutions (due to lower temperatures).